Project Summary/Abstract Nervous system function is highly dependent on the dynamic regulation of translation. Neural circuit assembly requires translation in axons as growth cones navigate to their targets and synaptic plasticity, the cellular basis of learning and memory, requires the activity-dependent translation of synaptically localized mRNAs. Once thought of as ubiquitous adaptors, tRNAs are emerging as key regulatory molecules in the dynamic regulation of translation. tRNA stability, efficiency and fidelity are controlled by extensive posttranscriptional modification. In a recent screen, we identified Drosophila tRNA methyltransferase 9-like (TRM9L) as a regulator of synaptic growth and neurotransmitter release. TRM9L is one of two animal paralogs of yeast TRM9, which methylates uridines in the wobble position of tRNA anticodon loops to modulate tRNA interactions with cognate vs. wobble codons and regulate the dynamic translation of stress response genes enriched for specific codons. Biochemical and genetic studies demonstrate that the TRM9 paralog ALKBH8 methylate wobble uridines. In contrast, TRM9L has remained biochemically uncharacterized in any system. With the generation of the first TRM9L loss-of-function model, we have identified a role for TRM9L in modifying tRNA wobble uridines in collaboration with the laboratory of Dragony Fu. Here, we propose experiments to build on our findings to gain an integrated understanding of TRM9L's role in the nervous system. In Aim 1, we combine genetic and imaging studies with biochemical analysis of tRNA modification in informative genetic backgrounds to functionally dissect TRM9L's role at synapses. In Aim 2, we will build on our finding that TRM9L also plays a conserved role in the response to oxidative stress. Interestingly, a number of tRNA modifying enzymes have recently been shown to play roles in both neurodevelopment and oxidative stress response, raising the possibility of mechanistic links. To investigate the relationship between TRM9L's roles at synapses and in oxidative stress resistance, we propose a comprehensive functional genetic analysis. In Aim 3, we propose computational and proteomic approaches to identify neuronal TRM9L target transcripts, followed by in vivo functional validation of top candidates. A growing list of links between tRNA modifications and neurological disorders underlines the importance of understanding the role of tRNA regulation in neuronal function. The proposed experiments will generate fundamental insight into the role of TRM9L in the nervous system and significantly expand our understanding of the dynamic regulation of protein expression and how its dysregulation alters nervous system function.